Wnt/2-catenin signaling is indispensable for osteoblastogenesis and bone formation. In studies leading to this application, a novel mechanism of Wnt/2-catenin antagonism, whereby activation of FoxO transcription factors diverts the limited pool of 2-catenin from Wnt induced TCF/Lef- transcription to FoxO-mediated transcription was elucidated. This antagonistic cascade in bone is initiated by reactive oxygen species (ROS) and represents both a mediator of, and a defense mechanism against, oxidative stress. FoxOs mediate oxidative stress responses by regulating the expression of genes involved in cell cycle, DNA repair, and lifespan. The involvement of the histone deacetylase Sirt1 in the inhibition of Wnt/2-catenin by ROS; and co- activation by 2-catenin of the expression of FoxO-target genes that promote osteoblast survival under stress conditions was also established. Moreover, evidence was obtained that the actions of glucocorticoids or TNF1 on bone are mediated, at least in part, by ROS-induced FoxO activation. The above observations form the foundation of the hypothesis that activation of FoxO transcription factors by oxidative stress represents a previously unappreciated cell-autonomous mechanism of Wnt/2-catenin antagonism which contributes to the adverse effects of aging, glucocorticoid excess and inflammatory cytokines on bone, by diverting 2-catenin from TCF- to FoxO-mediated transcription. To test this hypothesis, the role of ROS-induced FoxO post- translational modifications on the binding of FoxOs to 2-catenin, in osteoblastic cells, and whether ROS lead to recruitment of 2-catenin to the promoter of FoxO-target genes will be investigated (Aim 1). In addition, in vitro studies will be done to establish the consequences of FoxO activation in osteoblast lifespan and differentiation (Aim 2). Finally, mice overexpressing FoxO3a in osteoblast precursors and their progeny (osteoblasts, osteocytes); and mice in which the three main FoxO isoforms FoxO1, 3a, and 4 are conditionally deleted will be used to examine the role of FoxOs in skeletal homeostasis (Aim 3). This work should advance knowledge of how aging, glucocorticoid excess or inflammatory cytokines decrease bone mass. Furthermore, it should provide a better understanding of how to optimize the treatment of this condition. PUBLIC HEALTH RELEVANCE: The proposed studies seek to identify the means by which aging, glucocorticoid excess or inflammatory cytokines cause bone loss. This will be accomplished by studying changes in the function of proteins that control bone-forming cells. Increased understanding of the mechanisms that control bone formation will provide important information for the development of therapies to maintain or increase bone mass and strength, thereby reducing the risk of osteoporotic fractures